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PubMed2006

Methodologies to study implantation in mice.

Pregnancy begins with fertilization of the ovulated oocyte by the sperm. After fertilization, the egg undergoes time-dependent mitotic division while trying to reach the blastocyst stage and the uterus for implantation. Uterine preparation for implantation is regulated by coordinated secretions and functions of ovarian sex steroids. The first sign of contact between the blastocyst and the uterus can be detected experimentally by an intravenous blue dye injection as early as the end of day 4 or the beginning of day 5 of pregnancy. This blastocyst-uterine attachment reaction leads to stromal decidual reaction only at sites of implantation. The process of implantation can be postponed and reinstated experimentally by manipulating ovarian estrogen secretion. Stromal decidualization can also be induced experimentally in the hormonally prepared uterus in response to stimuli other than the embryo. Fundamental biological questions surrounding these essential features of early pregnancy can be addressed through the application of various techniques and manipulation of this period of early pregnancy. This chapter describes the routine laboratory methodologies to study the events of early pregnancy, with special emphasis on the implantation process in mice.

Anesthesia↗
PubMed1981

Resumption of DNA synthesis during activation of delayed implanting mouse blastocysts.

Cell division ceases in mouse blastocysts during the extended dormant period associated with delayed implantation but resumes following activation of the embryos by administration of 17 beta-estradiol to the mother. To determine the temporal and spatial aspects of the resumption of DNA synthesis during activation, blastocysts were recovered from delayed implanting females at various intervals after an injection of 17 beta-estradiol, incubated with 3H-thymidine in vitro, and prepared for light microscopic autoradiography. Although less than 4% of the cells were labeled in delayed implanting embryos, the proportion of labeled cells increased soon after the administration of 17 beta-estradiol and reached a maximum of over 50% by 24 hours. This increase in labeling was not uniform in all regions of the embryo, i.e., the labeling index of the inner cell mass began a steady increase immediately after the injection of 17 beta-estradiol while labeling of the polar and proximal mural trophoblast remained depressed for 6 and 12 hours, respectively, and only then began to increase. No labeling was present over the distal mural trophoblast in delayed implanting or activated blastocysts although cytological changes characteristic of primary giant cell transformation were present in activated embryos. These results indicate that the resumption of DNA synthesis is part of the overall increase in metabolic activity associated with activation. Furthermore, the sequential pattern of resumption of synthesis suggests that the ICM may influence the initiation of DNA synthesis in the surrounding trophoblast.

Animals↗
PubMed1996

A phorbol ester that activates protein kinase C mimics the action of estradiol or epidermal growth factor for initiating embryo implantation in the delayed implanting hypophysectomized rat.

In rodents an action of estrogen is required for the initiation of implantation of the blastocyst into the endometrium of a progesterone-primed uterus. Thus removal of endogenous estrogen, either directly by ovariectomy or indirectly by hypophysectomy, prevents implantation in the pregnant rat. In the present study, delayed implanting hypophysectomized progesterone-primed rats were used to test the efficacy of cyclic adenosine 3',5'-monophosphate (cAMP), epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), which are agents that have been shown to mimic some uterine actions of estradiol, to initiate implantation. In confirmation of previous studies, EGF injected into the uterine lumen plus intravenously was effective at initiating implantation in all animals. IGF-I showed no such activity in this model system. Cyclic AMP, increased via direct activation of adenylyl cyclase by forskolin, or administration of sodium dibutyryl cAMP, did not initiate implantation. However, a ligand for protein kinase C (PKC), phorbol 12-myristate 13-acetate, was effective in augmenting the action of intrauterine EGF, or forskolin, for initiation of implantation. A phorbol ester that does not activate PKC was ineffective. The results provide circumstantial evidence for the requirement of PKC activity in the implantation initiating action of estrogens.

Animals↗
PubMed1981

Comparative studies of embryo metabolism in early pregnancy.

Embryo metabolism during early pregnancy has been investigated, mainly in the mouse and rabbit, using a variety of catabolic and anabolic parameters. The rate of metabolic activity of the conceptus continues to increase markedly with the approach of implantation. However, in species in which the embryo enters a period of delayed implantation the diapause is associated with relative metabolic quiescence. Mouse, rabbit, ovine and bovine preimplantation embryos can be cultured in a simple defined medium. Under such conditions energy substrates in the medium represent a major source of carbon for anabolism. Glucose carbon is incorporated into macromolecules during in-vitro culture of cleaving mouse embryos and implanting and diapausing mouse blastocysts. In particular, both acid-soluble glycogen and desmoglycogen are rapidly synthesized from glucose presumably to act as a source of energy at implantation. The pattern of glucose accumulation by cleaving sheep embryos and embryonic discs and early somites excised from sheep and cattle embryos between Days 14 and 18 or pregnancy is basically similar to that in the mouse except that very little glycogen is synthesized by ruminant embryos. During embryonic diapause in the tammar wallaby there is a cessation of cell division and glucose uptake appears to be relatively low. After removal of pouch young in the breeding season to reactivate embryo development, glucose accumulation by the tammar blastocyst increases. The increase is related to the start of blastocyst expansion. Furthermore, the pattern of glucose metabolism in the reactivated wallaby blastocyst is similar to that in ruminant species, approximately 30% of the glucose carbon being incorporated into macromolecules such as protein, lipid and nucleic acid but with little synthesis of glycogen.

Animals↗
PubMedDate not supplied

Separation of a 170-K delayed-implantation-associated protein with inhibitory activity on trophoblast outgrowth and RNA synthesis by mouse embryo.

PROBLEM: To screen the uterine protein responsible for embryonic dormancy associated with delayed implantation. METHOD: Uterine protein extracts and sera from mice in which delayed implantation had been induced and those from pregnant mice were separated by three steps of chromatography and SDS-PAGE by monitoring an inhibitory activity on trophoblast outgrowth. The presence of the separated protein in the uterine luminal fluid was assessed. Effect of the protein on cell proliferation and RNA synthesis by blastocysts were assessed. RESULTS: A 170-K protein was found in the uterine tissue as well as uterine luminal fluid associated with delayed implantation. The 170-K protein suppressed RNA synthesis by approximately 50% and cell proliferation in blastocysts. CONCLUSION: A 170-K protein is secreted into the uterine lumen during delayed implantation period. The ability of 170-K protein to suppress RNA synthesis and cell proliferation may play a role in regulation of embryonic dormancy associated with delayed implantation.

Animals↗
PubMed2006

Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: possible origins and developmental significance of differential DeltaPsim.

Studies of mitochondria in mouse and human oocytes and preimplantation stage embryos have focused primarily on their metabolic capacity to generate ATP. However, it is becoming increasingly apparent that mitochondria are also regulatory agents in other processes involved in the establishment of developmental competence, including calcium homeostasis and apoptosis. The magnitude of the inner mitochondrial membrane potential, or its polarity (DeltaPsim), is a physiochemical property of mitochondria related to levels of organelle activity, and differences in the magnitude and spatial distribution of high- and low-polarized mitochondria have been suggested to influence oocyte and early embryo competence. Here, we investigated mitochondrial polarity in normal and diapausing peri-implantation-stage mouse blastocysts, and their corresponding outgrowths, for indications of cell-type-specific regulatory functions or activities in which these organelles may be engaged. The results demonstrate that cell-type- and location-specific domains of differential DeltaPsim exist in the peri-implantation blastocyst and remain unchanged during blastocyst outgrowth and during delayed implantation, which for the latter, is accompanied by the suppression of mitochondrial oxidative phosphorylation. Our findings demonstrate that cell-type-specific DeltaPsim in the peri-implantation blastocyst is not an intrinsic property of the corresponding mitochondria but one that can be mediated by the dynamics of intercellular contact. Cells with high- or low-polarized mitochondria are differentially affected by photosensitization, with developmental consequences related to embryo behavior and outgrowth performance. Differences in polarity are discussed with respect to the participation of mitochondria in regulatory and morphogenetic processes in the normal peri-implantation embryo. The persistence of high DeltaPsim in the diapausing embryo is suggested to be associated with the regulation of levels of cytoplasmic free calcium and the ability of the embryo to reactivate development when delayed implantation terminates.

Adenosine Triphosphate↗
PubMed2000

The toxic effects of Coleus barbatus B. on the different periods of pregnancy in rats.

Extracts of Coleus barbatus B. have been used in folk medicine to interrupt pregnancy. In order to evaluate if this plant interferes with embryo implantation or with the normal development of the concepts, pregnant Wistar rats were treated with increasing doses (220, 440 and 880 mg/kg per day) of a hydroalcoholic extract of C. barbatus. The rats received the extract by gavage from days 0 to 5 of pregnancy (preimplantation period) or 6 to 15 (organogenic period). Control groups received distilled water during the same periods. The animals were killed at term for the evaluation of maternal and fetal parameters. The results showed that the treatment with 880 mg/kg per day of the extract of C. barbatus before embryo implantation caused delayed fetal development and an anti-implantation effect, which justifies the popular use of this extract with abortive purposes. After embryo implantation delayed development associated with maternal toxicity was observed in the fetuses of the group which received 880 mg/kg per day.

Abortifacient Agents↗
PubMed1990

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes.

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.

Animals↗
PubMed1978

Factors in mouse uterine fluid that inhibit the incorporation of [3H]uridine by blastocysts in vitro.

Fluid flushed from the uteri of "delayed implanting" and "implanting" mice was fractionated by gel-filtration. The fractions were freeze-dried, individually resuspended in culture medium containing [3H]uridine, and used for incubating "implanting" blastocysts. Several factors were found that reduced incorporation of [3H]uridine by the blastocysts. The amount of inhibitory activity in corresponding factors was generally similar in flushings from both types of uteri, but there was significantly more inhibitory activity in the void volume fractions of flushings from "delayed implanting" animals and it is suggested that this factor may be responsible for the metabolic dormancy of embryos during the diapause associated with delayed implantation.

Animals↗
PubMed1999

Potential regulation of membrane trafficking by estrogen receptor alpha via induction of rab11 in uterine glands during implantation.

The steroid hormone estrogen profoundly influences the early events in the uterus leading to embryo implantation. It is thought that estrogen triggers the expression of a unique set of genes in the preimplantation endometrium that in turn control implantation. To identify these estrogen-induced genes, we used a delayed implantation model system in which embryo attachment to endometrium is dependent on estrogen administration. Using a mRNA differential display (DD) method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of estrogen. We identified one of these cDNAs as that encoding rab11, a p21ras-like GTP-binding protein (G protein), which functions in the targeting of transport vesicles to the plasma membrane. In normal pregnant rats, rab11 mRNA was expressed at low levels on days 1-2 of pregnancy, but its expression was markedly enhanced (approximately 6- to 8-fold) between days 3-5 immediately before implantation. In situ hybridization and immunocytochemistry revealed that rab11 expression in the uterus was predominantly in the glandular epithelium. In ovariectomized rats, the expression of rab11 mRNA was induced in the endometrium in response to estrogen. To determine whether this effect of estrogen was mediated through its nuclear receptors, we examined rab11 expression in a transformed endometrial cell line, Ishikawa. In transient transfection experiments, we observed that overexpression of estrogen receptor (ER) alpha or beta induced endogenous rab11 mRNA in a hormone-dependent manner. ER bound to an antagonist, ICI 182,780, failed to activate this gene expression. These findings, together with the observation that ER alpha but not ER beta is detected in the glands of the preimplantation uterus, indicate that rab11 is one of the proteins that are specifically induced by estrogen-complexed ER alpha in rat endometrium at the onset of implantation. Our results imply that estrogen, which induces the synthesis of many growth factors and their receptors and other secretory proteins that are thought to be critical for implantation, may also facilitate their transport to the membrane and/or secretion by stimulating the expression of rab11, a component of the membrane-trafficking pathway. This study therefore provides novel insights into the diverse cellular mechanisms by which estrogen, acting via its nuclear receptors, may influence blastocyst implantation.

Animals↗
PubMed1977

The culture of mouse blastocysts in the presence of uterine flushings collected during normal pregnancy, delayed implantation and pro-oestrus.

When day-3 mouse embryos were cultured in a simple medium supplemented with uterine fluids of mice autopsied on day 4 of pregnancy, 48 h after administration of oestradiol, or during pro-oestrus, the percentage of blastocysts hatching from the zona pellucida was significantly greater than in unsupplemented medium. In the presence of uterine fluids recovered during delayed implantation this stimulation of blastocyst hatching was not observed. When the culture medium was supplemented with dialysed uterine flushings containing 20 or 30 microgram protein/ml, both 'day 4' and 'delay' uterine proteins were equally effective in enhancing hatching frequency (P less than 0.05). The results suggested that 'delay' uterine fluids may contain a dialysable inhibitor of blastocyst activity. The putative inhibitor was not effective in the presence of serum, since uterine fluids recovered both on day 4 of pregnancy and during delayed implantation significantly increased the size attained by blastocyst outgrowths in the presence of foetal calf serum (P less than 0.01). The percentage of blastocysts exhibitng giant cell transformation and outgrowth was also increased (P less than 0.02) by these uterine fluids when the concentration of FCS in the medium was minimal (0.25%).

Animals↗
PubMed1996

Mechanisms of cell number regulation in the peri-implantation mouse blastocyst.

Low viability of manipulated or in vitro cultured is caused primarily by the reduced cell number in the implanting blastocysts. In order to investigate the effect of implantation delay on embryo viability and cell number, mouse blastocysts were transferred into oviducts of day 0 pseudopregnant females. This type of transfer improved embryo survival rates, indicating that embryos retarded by in vitro culture restored their viability during 3 days of delayed implantation. Our results showed that even in the cases when the initial cell count was as low as 28.2 +/- 0.7 cells per blastocyst (vs 60.5 +/- 1.4 cells in the control blastocysts, developed in vivo), implantation delay increased this number to 107.2 +/- 3.5 cells (control blastocysts had at this stage on average 111.0 +/- 3.7 cells). Half-blastocysts, developed from the single blastomeres of the 2-cell embryos or from experimentally produced tetraploids, had around 50 cells after 3 days of implantation delay. This indicates that the start of blastocyst dormancy is triggered during the eighth cell cycle and independent of the absolute cell number or the number cytokineses. Implantation-delayed blastocysts, developed from the half-embryos with the doubled volume of cytoplasm, had on average 70.5 +/- 2.4 cells, suggesting that embryo fall into quiescence is also dependent upon the attainment of a definite nucleo-cytoplasmic ratio. We conclude that blastocyst readiness for implantation is determined by two factors: number of cell cycles and nucleo-cytoplasmic ratio.

Animals↗
PubMed1989

Early pregnancy wastage following late implantation of embryos after in-vitro fertilization and embryo transfer.

Serial plasma concentrations of human chorionic gonadotrophin (HCG), progesterone and oestradiol were measured in pregnancies after in-vitro fertilization and embryo transfer. The first detection day of HCG after embryo transfer (8.4 +/- 1.1) and the HCG doubling time (DT) of 64 normal singleton pregnancies were compared to those of 14 first-trimester miscarriages. The same parameters were evaluated in nine late-implanted conceptions, seven of which resulted in early pregnancy wastage. The HCG DT of late-implanted pregnancies was consistent with that of singleton term pregnancies in the first 12 days, while first-trimester miscarriages which had implanted at the usual time had a significantly longer DT from implantation onwards. The reduced trophoblastic secretory rate suggests poor embryo quality in these cases. A decreased progesterone/oestradiol ratio was observed in late-implanted pregnancies but because of the small number of individuals, no definite conclusion can be drawn. More patients with delayed implantation should be tested to justify this observation.

Adult↗
PubMed1986

Chlorpromazine inhibits the mitotic index, cell number, and formation of mouse blastocysts, and delays implantation of CBA mouse embryos.

Chlorpromazine, administered to pregnant CBA mice 56 h after copulation in single doses of 10 or 15 mg/kg bodyweight, inhibited the compaction of embryos, formation of blastocysts, and reduced the mitotic index and cell number of embryos 86 h after copulation but did not adversely influence their viability or induce structural chromosomal aberrations. Blastocyst formation was more severely affected than embryo compaction. When 86-h embryos were treated with chlorpromazine (10 or 15 mg/kg) and subsequently cultured for 120 h, there was delayed hatching from the zona pellucida, delayed attachment to the culture dish, outgrowth of the trophoblast and expansion of the inner cell mass. Mice treated identically and evaluated on the 18th day of gestation had fewer implanted embryos than did controls, and the fetuses weighed less. No resorptions, malformations or significant differences in intrauterine deaths were found. Chlorpromazine given in the same manner but at 0.5 mg/kg did not affect any of the aforementioned criteria. When 56 h embryos were cultured in vitro in the presence of 50 microM-chlorpromazine for a further 40 h, embryo compaction, blastocyst formation, the mitotic index and the total cell number were significantly reduced compared with controls. Blastocyst formation was again more severely affected than embryo compaction. The inhibition of embryo compaction, blastocyst formation, and reduction in mitotic index and cell number associated in this study with chlorpromazine in vivo and in vitro indicate that the drug inhibits the development of cleavage-stage embryos in the mouse. These effects might be mediated by antagonistic effects of calmodulin.

Animals↗